The present invention relates generally to forms of microbial xcex2-glucuronidase that are directed to specific cell compartments, and more specifically to a secreted form of xcex2-glucuronidase and uses of these xcex2-glucuronidases thereof.
The natural habitat of E. coli is the gut, and the xcex2-glucuronidase (GUS) activity of E. coli plays a specific and very important role in its natural history. The gut is a rich source of glucuronic acid compounds, providing a carbon source that can be efficiently exploited by E. coli. Glucuronide substrates are taken up by E. coli via a specific transporter, the glucuronide permease (U.S. Pat. Nos. 5,288,463 and 5,432,081), and cleaved by xcex2-glucuronidase. The glucuronic acid residue thus released is used as a carbon source. In general, the aglycon component of the glucuronide substrate is not used by E. coli and passes back across the bacterial membrane into the gut to be reabsorbed into the bloodstream and undergo glucuronidation in the liver, which begins the cycle again.
In E. coli, xcex2-glucuronidase is encoded by the gusA gene (Novel and Novel Mol. Gen. Genet. 120:319-335, 1973), which is one member of an operon comprising three protein-encoding genes. The second gene, gusB, encodes a specific permease (PER) for xcex2-glucuronides. The third gene, gusC, encodes an outer membrane protein (MOP) of approximately 50 kDa that facilitates access of glucuronides to the permease located in the inner membrane. The principle repressor for the GUS operon, gusR, maps immediately upstream of the operon.
xcex2-glucuronidase activity is expressed in almost all tissues of all vertebrates and many mollusks (Levvy and Conchie, 1966). In addition, the free-living soil nematode, Caenorhabditis elegans, has an endogenous xcex2-glucuronidase activity (Sebastiani et al, 1987; Jefferson et al, 1987), which occurs at low levels in the intestine of the worm. The enzyme has been purified from many mammalian sources (e.g. Tomino et al, 1975) and forms a homotetrameric structure with a subunit molecular weight of approximately 70 kDa.
The vertebrate enzyme is synthesized with a signal sequence at the amino terminus, then transported to and glycosylated within the endoplasmic reticulum, and ultimately localized intracellularly within vacuoles. If any of the mammalian enzyme is secreted, it probably contributes little to the total activity as the enzyme is relatively unstable. Thus, for use in medical diagnostics (e.g., drug testing) and transgenic constructions, the E. coli enzyme is preferred because it is much more active and stable than the mammalian enzyme against most biosynthetically derived xcex2-glucuronides (Tomasic and Keglevic, 1973; Levvy and Conchie, 1966).
Production of GUS for use in in vitro assays, such as medical diagnostics, is costly and requires extensive manipulation as GUS must be recovered from cell lysates. A secreted form of GUS would reduce manufacturing expenses, however, attempts to cause secretion have been unsuccessful. In addition, for use in transgenics, the current GUS system has somewhat limited utility because enzymatic activity is detected intracellularly by deposition of toxic calorimetric products during the staining or detection of GUS. Moreover, in cells that do not express a glucuronide permease, the cells must be permeabilized or sectioned for introduction of the substrate. Thus, this conventional staining procedure generally results in the destruction of the stained cells. In light of this limitation, a secreted GUS would allow for development of non-destructive marker system, especially useful for agricultural field work.
The present invention provides gene and protein sequences of secreted xcex2-glucuronide, variants thereof, and use of the protein as a transformation marker, while providing other related advantages.
In one aspect, an isolated nucleic acid molecule is provided comprising a nucleic acid sequence encoding a secreted form of xcex2-glucuronidase, wherein the nucleic acid sequence comprises the amino acid sequence as presented in FIG. 3 SEQ ID No: 2 or hybridizes under stringent conditions to the complement of the sequence comprising nucleotides 1662-3467 of FIG. 1 SEQ ID No: 1 and which encodes a functional xcex2-glucuronidase. In preferred embodiments, the nucleic acid molecule comprises nucleotides 1662-3467 of FIG. 1 SEQ ID No: 1 or encodes the amino acid sequence of FIG. 3, SEQ ID No: 2 or a variant thereof.
In another aspect, the invention provides an isolated secreted form of xcex2-glucuronidase, wherein xcex2-glucuronidase is encoded by the isolated nucleic acid molecule or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 SEQ ID No: 1 and which encodes a functional xcex2-glucuronidase. In a preferred embodiment, the isolated secreted form of xcex2-glucuronidase comprises the amino acid sequence of FIG. 3, SEQ ID No: 2 or a variant thereof.
The invention also provides vectors and host cells, comprising a nucleic acid molecule encoding a secreted form of xcex2-glucuronidase, wherein the xcex2-glucuronidase sequence is in operative linkage with a promoter element. In preferred embodiments, the promoter element is a promoter derived from a plant pathogen. Preferred host cells are selected from the group consisting of a plant cell, an insect cell, a fungal cell, an animal cell and a bacterial cell.
The invention also provides a method of producing a secreted form of xcex2-glucuronidase, comprising: (a) introducing a vector comprising a nucleic acid molecule encoding a microbial xcex2-glucoronidase into a host cell, wherein the vector comprises nucleic acid sequence encoding the xcex2-glucuronidase is expressed. The method may further comprise isolating the xcex2-glucuronidase from cell supernatant or periplasm.
In other aspects, the invention provides methods of introducing a controller element into a host cell, monitoring expression of a gene of interest or a portion thereof in a host cell, monitoring activity of a controller element in a host cell, transforming a host cell with a gene of interest or portion thereof, and positive selection for a transformed cell.
In other aspects, transgenic cells are provided, such as plant cells, insect cells, and transgenic plants and insects.
In other aspects, kits comprising microbial GUS are provided.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.